Method for preparing 4&#39;-Demethylepipodophyllotoxin from podophyllotoxin

ABSTRACT

A method for synthesizing a 4&#39;-demethylepipodophyllotoxin of formula (II) from a podophyllotoxin of formula (I) by treating it with a pair of reagents, i.e. a strong acid and an aliphatic, aromatic or functionalized sulphide, in the present of an organic or inorganic acid, or in the presence of water with or without a water-miscible organic solvent. ##STR1##

The present application is a U.S. National Application filed under 35USC 371 of PCT/FR96/02000, filed Dec. 13, 1996 based upon Frenchapplication Serial No. 95/14875 filed Dec. 14, 1995.

The present invention relates to a novel method for the demethylation ofpodophyllotoxin I in order to access 4'-demethylepipodophyllotoxin offormula II ##STR2##

4'-Demethylepipodophyllotoxin is a key synthetic intermediate in thepreparation of novel anticancer substances, of which mention may bemade, for example, of etoposide and teniposide.

The preparation of 4'-demethylepipodophyllotoxin II is known and isusually carried out by demethylation of podophyllotoxin I by HBr,according to the original method described by M. Kuhn, Helvetica ChimicaActa, 1969, 52, 944, and then taken up by several groups, such as K. H.Lee, J. Med. Chem., 1986, 29, 1547 and Journal of Natural Products,1989, 52, 606, O. Buchardt, Acta Chemica Scandinavica, 1993, 47, 1190 orY. Nishimura, Chemistry Letters, 1987, 799. This method gives moderateyields, according to the amounts involved, and is relatively difficultto carry out, because of the handling of highly aggressive gaseous HBr.Other methods can be envisaged for carrying out this demethylation, byusing the usual reagents for such a conversion: mention may be made ofBBr₃ or BCl₃ in CH₂ Cl₂ from -20° C. to +20° C. (Tet., 1969, 24, 2289);AlCl₃ in CH₂ Cl₂ at 0° C. or at ordinary temperature (according to J.Chem. Soc., 1944, 330); pyridinium hydrochloride at more or less hightemperature; AlBr₃ in the presence of ethanethiol at ordinarytemperature.

These various methods result in the degradation or in the non-conversionof podophyllotoxin.

A novel demethylation method has therefore been developed.

The method which is the subject-matter of the present invention consistsin treating podophyllotoxin I with the pair of reagents: strongacid-aliphatic, aromatic or functionalized sulfide, in the presence ofan organic or inorganic acid or else in the presence of water, with orwithout water-miscible organic solvent, such as the acetone-watermixture, advantageously, at a temperature of between -20° C. and +40° C.

According to the specific characteristics of the present invention, thestrong acid of the pair of reagents is advantageously composed ofmethanesulfonic acid, whereas the aliphatic, aromatic or functionalizedsulfide of the pair of reagents is advantageously chosen from dimethylsulfides D,L-methionine and methylthioacetic acid.

According to other advantageous characteristics of the method of theinvention, the reaction is carried out in the presence of an organicacid of formula RCOOH, where R=H, C₁ -C₄ alkyl or CX₃, with X=Cl or F.It will relate in particular to formic acid or trifluoroacetic acid.

In the case where the reaction is carried out in the presence of aninorganic acid, use can advantageously be made of H₃ PO₄ or a slightstream of HCl or HBr sparging into the reaction mixture.

In accordance with the present invention, the compound II directlyresulting from the reaction is isolated and purified byrecrystallization from a solvent, such as isopropyl alcohol, aceticacid, water, acetone, dioxane, isopropyl ether, toluene, ethanol andtheir mixture.

D,L-Methionine can also be replaced by an aliphatic or aromatic sulfidewith a low carbon number or with a functionalized sulfide, for examplefunctionalized by an acidic functional group, such as methylthioaceticacid. In this case, the joint presence of an inorganic or organic acidis no longer necessarily indispensable.

It is known in the chemical literature that the methanesulfonic acid,D,L-methionine pair can demethylate the aromatic methoxy of certaincompounds, in order to result in the corresponding phenols: see J. C.S., Perkin Trans. I, 1977, 2288; J. C. S. Chem. Comm., 1976, 922; Chem.Lett., 1980, 875; J. Med. Chem., 1984, 27, 28; Heterocycles, 1992, 34,937; Synth. Comm., 1992, 22 (16), 2313. One of the reaction products isthus the methylsulfonium derivative of methionine, which means that analiphatic or aryl sulfide can also be used as demethylation reagent,resulting in the corresponding sulfonium: see, in this respect: M.Julia, Tet. Let., 1979, No. 13, 1101.

It appears that this demethylation method, applied to podophyllotoxin I,only results in decomposition products and consequently cannot be used.

In contrast, if the operation is carried out in the presence of anorganic acid as indicated above and in particular formic acid, aceticacid or trifluoroacetic acid or in the presence of an inorganic acid, inparticular phosphoric acid, or in the presence of a water-miscibleorganic solvent, such as the acetone-water mixture, it is possible toselectively demethylate the methoxy in the 4'-position and to obtain thedesired product with a good yield.

The inorganic acid can also be gaseous hydrochloric or hydrobromic acid;it is thus possible to use this method, methanesulfonic acid,D,L-methionine on podopyllotoxin I, where the alcohol functional groupin the 4-position would have been easily converted beforehand under mildconditions, by virtue of this slight stream of hydrochloric orhydrobromic gas, to halide, Cl or Br, these operations being carried outin a "one pot" process, with the treatment with methanesulfonic acid andmethionine; these processes are described in the examples hereinbelow.This operation of gentle treatment with gaseous Hbr is not capable ofdemethylating the aromatic methoxy in the 4'-position and only uses avery small amount of gas and for a very short time, with respect to theprior process.

The advantage of this method is its simplicity of operation and thepurity of the reaction mixture obtained with respect to the priormethod, not requiring the handling of large amounts of aggressive gasesand specific equipment.

The yields of this demethylation to 4'-demethylepipodophyllotoxin areimproved with respect to the prior art. This process can be applied onan industrial scale without particular difficulties by increasing theamounts of substance involved, without loss of yield.

Furthermore, an additional improvement in the process lies in the factthat it is not necessary to purify the compound obtained by columnchromatography, which is a lengthy and expensive operation. It ispossible, at the end of the reaction, to precipitate the product formedfrom water and ice, in order to obtain a crystalline product which canbe filtered off and recrystallized from solvents, such as, for example,isopropanol, isopropyl ether, acetone, acetic acid or water and theirmixture. It is also possible, at the end of the reaction, to extract theproduct with normal solvents to crystallize and recrystallize theevaporation residue from the abovementioned solvents. The reactionproduct can be used crude, even without recrystallization, in subsequentsynthetic stages, such as, for example, the protection of the phenol inthe 4'-position by benzyloxycarbonyl chloride.

The following examples illustrate the various procedures used for thisdemethylation:

    ______________________________________                                        Ex-   Starting                                                                  ample material Reagents Temperature Time Yield                              ______________________________________                                        1     1      5 g   CH.sub.3 SO.sub.3 H(20 eq)                                                              Ordinary                                                                              --    Degrad-                                 D,L-methionine temperature  ation                                             (1.1 eq)                                                                   2 1  12 g CH.sub.3 SO.sub.3 H (45 eq) 40° C. 0.75 h 93%                   D,L-methionine                                                                (5 eq)                                                                        CF.sub.3 CO.sub.2 H (6 eq)                                                 2a 1 100 g CH.sub.3 SO.sub.3 H (45 eq) 0° C. to   1 h 65%                                                           D,L-methionine 10-20.degree                                               . C.                                    (5.5 eq)                                                                      CF.sub.3 CO.sub.2 H (10 eq)                                                3 1  7 g CH.sub.3 SO.sub.3 H (37 eq) 40° C. 25 min Crystal-                                                         D,L-methionine   lized                                                        (8 eq)   yield:                     HCO.sub.2 H (4.5 eq)   48%                                                       Chrom-                                                                        ato                                                                           yield:                                                                        48%                                                                     4 1  5 g CH.sub.3 SO.sub.3 H (50 eq) 0° C., then   6 h 70%                                                          D,L-methionine return to                                                      (6 eq) ordinary                     H.sub.3 PO.sub.4 temperature                                               5 1  1 g CH.sub.3 SO.sub.3 H (32 eq) -10° C.     1 h 52%                                                            D,L-methionine                      (1.5 eq)                                                                      HCl gas                                                                    6 1  50 g CH.sub.3 SO.sub.3 H (64 eq) 40° C. and   2 h 80%                                                          D,L-methionine return to                                                      (5.5 eq) ordinary                   Acetone-water temperature                                                     5/1                                                                        7 1  5 g CH.sub.3 SO.sub.3 H (20 eq) -10° C.     2 h 54%                                                            D,L-methionine                      (2 eq)                                                                        Hbr gas                                                                  ______________________________________                                    

Preparation of 4'-demethylepipodophyllotoxin II

The podophyllotoxin used has a natural origin and exhibits a purity of95%.

EXAMPLE 1

According to H. Yajima, J. C. S., Perkin I, 1977, 2288

1 g (2.4 mmol) of podophyllotoxin I are reacted with stirring with 400mg (2.64 mmol) of D,L-methionine and 3.1 ml (48 mmol) of methanesulfonicacid at ordinary temperature. The mixture immediately turns dark red incolor. The degradation of the starting material is observed by TLC.

EXAMPLE 2

Use of Trifluoroacetic Acid

12 g (29 mmol) of podophyllotoxin I are dissolved in 20 ml oftrifluoroacetic acid and stirred at room temperature. 21.6 g (0.14 mol)of D,L-methionine and 84 ml (1.29 mol) of methanesulfonic acid areintroduced, the temperature rises to 40° C. and stirring is maintainedfor 45 min. The reaction mixture is then plunged into 500 ml of amixture of equal volumes of acetone and ice-cold water. Neutralizationis carried out with stirring to pH 4 to 5 by addition of K₂ CO₃ and thenextraction is carried out twice with ethyl acetate, the organic phasesare dried over Na₂ SO₄ and filtered, and the solvent is evaporated underreduced pressure. The evaporation residue crystallizes from isopropylether, 10.7 g, Yd 93%.

The 4'-demethylepipodophyllotoxin obtained according to this process canafterwards be used directly in subsequent reactions, such as theprotection of the phenol in the 4'-position by benzyloxycarbonylchloride.

EXAMPLE 2a

Use of Trifluoroacetic Acid

100 g (0.24 mol) of podophyllotoxin I are dissolved in 186 ml oftrifluoroacetic acid with stirring. After cooling the reaction mixtureto 0° C., a solution of 198 g (1.32 mol) of D,L-methionine in 500 ml ofmethanesulfonic acid is introduced, the reaction mixture beingmaintained between 10 and 20° C. 200 ml of methanesulfonic acid areadded to the mixture, which is kept stirring for 1 h at thistemperature. The reaction mixture is poured with stirring onto 4 l ofwater and of ice and the product precipitates. It is extracted withethyl acetate (3×1 l ). The combined organic phases are washed with anNaHCO₃ solution and then dried over Na₂ SO₄, filtered and evaporatedunder reduced pressure in order to obtain 91.5 g (Yd 94%) of crude4'-demethylepipodophyllotoxin.

Although the product can be used directly in the following syntheticoperations, the product can be purified by chromatography (SiO₂ CH₂ Cl₂/acetone 95/5) to provide 65% of pure 4'-demethylepipodophyllotoxin. Thecrude reaction product can also be purified by recrystallization fromisopropyl alcohol, acetone, acetic acid or their mixture with water.

EXAMPLE 3

Use of Formic Acid

7 g (17 mmol) of podophyllotoxin I are dissolved in 3.5 ml of formicacid, one drop of methanesulfonic acid is added and the reaction mixtureis stirred for 15 min at ordinary temperature, and a clear pale-yellowsolution is obtained. 10.5 g (68 mmol) of D,L-methionine and 49 ml (0.63mol) of methanesulfonic acid are then introduced with stirring and thetemperature rises to approximately 400° C. over 10 min. 10.5 g ofD,L-methionine are again added to the reaction mixture. After 15 min,the mixture is plunged into 500 ml of water and of ice and a precipitateis obtained which is extracted 3 times with ethyl acetate, washed withan NaHCO₃ solution, dried over Na₂ SO₄ and evaporated in order to obtain5.5 g of the compound II (Yd 81%). The product obtained is crystallizedfrom the isopropyl ether and CH₂ Cl₂ mixture in order to obtain 3.2 g ofpure 4'-demethylepipodophyllotoxin (Yd 48%). Chromatography carried outon the crude reaction product (SiO₂ -elution CH₂ Cl₂ -90-acetone-10)provides exactly the same result (3.2 g of the derivative II, Yd 48%).

EXAMPLE 4

Use of Phosphoric Acid

5 g (12 mmol) of podophyllotoxin I are added in a single step to themixture consisting of phosphoric acid (2.5 ml), methanesulfonic acid (40ml, 0.6 mol) and D,L-methionine (11 g, 72 mmol) cooled to 0° C. withstirring which is maintained for 6 hours with return to ordinarytemperature.

The reaction mixture is then poured with stirring onto 1 l of water andof ice in order to obtain a white precipitate. It is then extracted withethyl acetate three times and then washed with an NaHCO₃ solution. Theorganic phases are combined, dried over Na₂ SO₄, filtered and evaporatedunder reduced pressure in order to obtain 4.2 g (Yd 88%) of4'-demethylepipodophyllotoxin II. The purity of the batch obtained is79% (HPLC analysis). The crude residue obtained can be recrystallizedfrom solvents such as the acetone water (5/2), toluene-ethanol (90/10)or dioxane-isopropyl ether (50/50) mixture.

EXAMPLE 5

Use of a 4-chloro-4-deoxyepipodophyllotoxin Intermediate

1 g of podophyllotoxin I is dissolved in a mixture of 15 ml of CH₂ Cl₂and 5 ml of ethyl ether. A slight stream of gaseous hydrochloric acid isintroduced into the solution for 15 min at -10° C. and then stirring ismaintained at this temperature for 1 h. After evaporating the reactionmixture without heating, 0.54 g of D,L-methionine and then 5 ml ofmethanesulfonic acid are added. After dissolving and stirring for 1 hwith return to ordinary temperature, the reaction mixture is plungedinto a water-acetone mixture and then BaCO₃ is added to neutrality.After extracting with ethyl acetate, separating by settling, drying overNa₂ SO₄, filtering and evaporating under reduced pressure, a residue isobtained which is crystallized from ethyl ether to provide 0.5 g (Yd52%) of white crystals of 4'-demethylepipodophyllotoxin exhibiting ahomogeneous spot by TLC.

EXAMPLE 6

Use of the Acetone-water Mixture

A solution of 50 g (0.12 mol) of podophyllotoxin I, prepared in 50 ml ofacetone, is added with stirring at ordinary temperature to a mixtureconsisting of 100 g (0.67 mol) of D,L-methionine, of 500 ml (7.7 mol) ofmethanesulfonic acid and of 10 ml of water. The temperature rises toapproximately 40° C. and stirring is maintained for 2 h with return toambient temperature. The mixture is then poured onto ice in order toobtain a precipitate which is extracted with ethyl acetate (3×700 ml).The organic phases are washed with an NaHCO₃ solution and then separatedby settling, dried over Na₂ SO₄, filtered and evaporated under reducedpressure in order to obtain 4'-demethylepipodophyllotoxin, existing inthe form of a white solid. 45 g (Yd 94%) are obtained.

This residue is taken up in isopropyl ether in order to obtain 38 g (Yd80%) of 4'-demethylepipodophyllotoxin. The product thus obtained canoptionally be further recrystallized from an acetone-water mixture butit can nevertheless be used directly in the subsequent synthetic stages.

EXAMPLE 7

Use of a 4-bromo-4-deoxyepipodophyllotoxin Intermediate

5 g of podophyllotoxin I are dissolved at -10° C. with slow stirring ina mixture of 100 ml of CH₂ Cl₂ and 35 ml of Et₂ O. A slight stream ofgaseous HBr is introduced into this solution for 10 min. At this stage,the compound obtained III (R=Br) is that described by M. Kuhn, HelveticaChimica Acta, 52, 944 (1969). The reaction mixture is then evaporatedunder reduced pressure without heating. 3.6 g (24 mmol) ofD,L-methionine and 15.5 ml (0.24 mol) of methanesulfonic acid aresubsequently introduced with stirring and the reaction is thusmaintained for 2 h at room temperature. The reaction mixture is thenintroduced dropwise into a mixture containing equal volumes of acetoneand water saturated with NaHCO₃, in order to neutralize. After stirringfor 10 min, water is added and extraction is carried out with ethylacetate, the mixture is separated by settling and the organic phases aredried over Na₂ SO₄, filtered and then evaporated under reduced pressurein order to obtain 4.7 g (98%) of crude 4'-demethylepipodophyllotoxin.Chromatography on silica (elution CH₂ Cl₂ -acetone 90-10) provides 2.6 gof pure 4'-demethylepipodophyllotoxin (Yd 54%).

We claim:
 1. Method for the synthesis of 4'-demethylepipodophyllotoxinof formula II from podophyllotoxin of formula I, which consists intreating the podophyllotoxin with a pair of reagents consisting ofmethanesulfonic acid, on the one hand, and dimethyl sulfide,D,L-methionine, or methylthioacetic acid, on the other hand, in thepresence of water, with or without the present of a water-miscibleorganic solvent ##STR3##
 2. Method according to claim 1, wherein thesynthesis is carried out at a temperature between about -10° C. and 40°C.
 3. Method according to claim 1 wherein the compound II directlyresulting from the reaction is isolated and purified byrecrystallization from a solvent.
 4. Method of claim 3 wherein thesolvent is selected from the group consisting of isopropyl alcohol,acetic acid, water, acetone, dioxane, isopropyl ether, toluene, ethanol,and mixtures thereof.
 5. Method according to claim 2, wherein thecompound II directly resulting from the reaction is isolated andpurified by recrystallization from a solvent.
 6. Method of claim 5wherein the solvent is selected from the group consisting of isopropylalcohol, acetic acid, water, acetone, dioxane, isopropyl ether, toluene,ethanol, and mixtures thereof.